Isolation of Two Isochlorogenic Acid Isomers from Phenolic Rich Fraction of Artemisia turanica Krasch.

Total phenolic content (TPC) and antioxidant capacity of five different extracts (petroleum ether (40-60), dichloromethane, ethyl acetate, ethanol and ethanol-water (1:1 v/v)) of Artemisia turanica (A. turanica) aerial parts were determined and phytochemical study on the most promising extract was carried out. Folin–Ciocalteu method, 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging test, β-carotene bleaching (BCB) method, and ferrous ion chelating (FIC) assay were performed. Vacuum liquid chromatography (VLC) and semi-preparative HPLC were used for bioassay-guided phytochemical isolation. Structures of isolated compounds were established using spectroscopic analysis including NMR and MS. Among all the extracts analyzed, the hydroethanolic extract exhibited the highest phenolic content and antioxidant activity. VLC of this extract yielded seven fractions (A to G) which were subjected to all antecedent experiments. The same sample (Fraction D) showed the highest total phenolic content and free radical scavenging activity but the only statistically significant correlation between TPC and EC50 values was observed for BCB. 3,5-dicaffeoylquinic acid (isochlorogenic acid A), and 4,5-dicaffeoylquinic acid (isochlorogenic acid C) was isolated from the most active fraction. Antioxidant activity of A. turanica is probably partly due to the presence of isomers of isochlorogenic acid.


Introduction
The genus Artemisia, belonging to tribe Anthemideae (family Asteraceae), contains the well-known medicinal plants. Different species could be found in the temperate zone of Asia, Europe, and North America (1). About 34 Artemisia species have been identified in Iran (2). A. turanica Krasch. (locally named "Dermaneye Ghermez") which grows naturally in the northeastern region of the country is one of these species (3). 1,8-cineole, chrysanthenone, and davanone have been reported as major constituents of the essential oil of the aerial parts while another study has shown a notable increase of α-thujone content in the leaf essential oil (4,5). Methanolic extract of the aerial parts of the plant has been effective against BacillusSubtilis, Staphylococcus aureus and Pseudomonas aeruginosa (6). Moderate toxicity of crude hydroethanolic extract against HepG2 cell line as well as the moderate effect of the ethanolic extract on in-vitro leishmanicidal activity have been reported (7,8). There are some reports on the antimalarial activity of A. turanica extracts as well (9)(10)(11). While the essential oil and different extracts of A. turanica have shown antioxidant and cytotoxic activity, a recent study has demonstrated the in-vitro neuroprotective potential of A. turanica (12)(13)(14). As per the previous reports enclosing the diverse beneficial effects of A. turanica, the authors have been stimulated to study the antioxidant effect of this species using different assays. To the best of our knowledge, although collectively these studies suggested the therapeutic benefits of this medicinal plant, there is no comprehensive study on the free radical scavenging and ferrous ion chelating activities of different extracts and fractions. This study aims to investigate the antioxidative role of different extracts/ fractions of A. turanica using different assays and examine whether their activity is correlated with the phenolic content. Another aim of the present work is to determine the structure of phytochemicals which are responsible for the observed effects.

Preparation of extracts
Air-dried and ground aerial parts (200 g) of A. turanica were extracted with petroleum ether (40-60), dichloromethane, ethyl acetate, ethanol and ethanol-water (1:1 v/v), respectively (Sequential maceration with ca. 3 × 2 L of each solvent). The extracts were filtrated with filter paper and dried using rotary evaporator at reduced pressure at a temperature below 45 °C to yield 5.51, 24.23, 1.21, 7.78, and 37.50 g of each extract, respectively.

Chromatographic Fractionation and Isolation
Reversed-phase high performance liquid chromatography (RP-HPLC) is now commonly used for the separation of complex the mixtures of phenolic compounds and the other natural products (15). Fifteen grams of the hydroethanolic extract which exhibited better results in two different antioxidant assays was subjected to reversed-phase VLC using a step gradient of MeOH-H 2 O (0.5:9.5, 1:9, 2:8, 4:6, 6:4, 8:2, 10:0) to give seven fractions (A, B, C, D, E, F and G) respectively (

Total phenolic contents
The total phenolic content (TPC) was measured by the Folin-Ciocalteu method with some modifications (16,17). Different concentrations of samples in water (0.500 mL) were mixed with 2.5 mL of Folin-Ciocalteu reagent (0.2 N). Two mLof Na 2 CO 3 solution (75 g/L) was added after 5 min. After 2 h standing in the dark, the optical density was measured at 760 nm against a blank. The total phenolic contents were calculated based on the calibration curve of gallic acid and expressed as milligrams of gallic acid equivalents (GAE), per gram of the dried samples.

DPPH radical scavenging activity
The assay was performed according to the method of Hatano et al. with slight modifications (18). Briefly, test samples were dissolved in methanol at different concentrations. Equal volumes of 0.2 mM solution of DPPH in methanol were added to each of the test tubes. The mixture was shaken vigorously and maintained in the dark for 30 min. Then, the absorbance was read at 517 nm against a blank. Butylated hydroxytoluene (BHT) and ascorbic acid were used as standard references. The scavenging activity was calculated using the formula: Where A c = absorbance of the control and A = absorbance of a tested sample in 30 min.

Metal chelating activity
The chelating activity of the extracts and fractions for ferrous ions Fe 2+ was measured adopting the ferrous iron-ferrozine complex

Inhibition of β-carotene bleaching
Antioxidant potential of the extracts and fractions was determined by a slightly modified version of the β-carotene bleaching method (20). Linoleic acid (33 µL) was added to 225 mg of Tween 40 and 750 µL of β-carotene solution (0.500 mg/mL). The solvent was completely removed using a rotary evaporator. After adding 75 mL of oxygenated distilled water, the mixture was emulsified for 15 min in a sonicator to give emulsion A. Aliquots of 3.5 mL of this emulsion were transferred into a series of stopper test tubes containing 1 mL of the samples dissolved in water or DMSO in different concentrations. Optical density (OD) at 470 nm was recorded for all the samples immediately (t = 0) and at the end of the assay time (t = 120). An emulsion which consisted of 50 mL of oxygenated water, 22 µL of linoleic acid and 150 mg of Tween 40 was also prepared to be used as the blank to zero the spectrophotometer. The percentage of inhibition was calculated according to the following formula: I% = (A A (120) -A C (120) )/(A C (0) -A C (120) ) × 100 Where A A (120) is the absorbance of the sample at t = 120 min, A C (120) is the absorbance of the control at t = 120 min, and A C (0) is the absorbance of the control at t = 0 min.

Statistical analysis
All the experiments were performed in triplicate. The data were reported as mean ± standard deviation (SD) (n = 3) and evaluated by non-parametric Friedman test. The difference was considered to be statistically significant if P < 0.05. Pearson's correlation coefficients (r) between the total phenolic contents of the samples and calculated EC 50 values were determined in each antioxidant assay.

Total Phenolic Content
Regression equation of the calibration curve of gallic acid (R 2 = 0.997, y = 0.011x + 0.057) was used to calculate the content of phenolics and expressed in GAE as milligrams per gram of each sample (mg GAE/g extract or fraction). Large variations in TPC of the samples were found, ranging from 24.36 ± 1.55 (fraction A) to 255.00 ± 10.29 (fraction D) mg GAE/g fraction (Table 1).

DPPH radical scavenging activity
All the samples except fraction A and three extracts (petroleum ether, dichloromethane, and ethyl acetate) showed moderate to strong scavenging activity on the DPPH radical. The highest activity was recorded for ethanolic extract, with the EC 50 value of 18.31 ± 0.59 µg/mL, followed by the fraction F and hydroethanolic extract with the EC 50 values of 18.43 ± 0.45 and 20.13 ± 1.07 µg/mL, respectively (Table 1).

Metal chelating activity
The highest ferrous ion chelating effect among the samples was shown by fraction B, with the EC 50 value of 28.96 ± 3.23 µg/ mL followed by hydroethanolic extract and fraction A with the EC 50 values of 47.88 ± 4.72 µg/mL and 47.92 ± 19.35 µg/mL, respectively (Table 1). While fractions C to G had moderate activity, the other extracts did not show any remarkable color changes, although decreases in absorbance readings-except petroleum ether extract-were recorded.

Statistical analysis
Pearson's correlation coefficients between TPC and calculated EC 50 values for DPPH, FIC and BCB assays took the values of -0.531, -0.032, and -0.696, respectively. The lowest correlation was seen between the TPC of the samples and their ability to chelate ferrous ions. No significant correlation was observed between TPC and DPPH radical scavenging activities of the samples as well. The highest correlation between the results of BCB assay and total phenolic contents was observed. The results of Friedman test showed no significant difference in the assays in screening the samples for their antioxidant ability.

Discussion
To the best of our knowledge, this is the first report on the presence of two dicaffeoylquinic acid isomers (isochlorogenic acids A and C) in A. turanica. The structures of the isolated compounds were elucidated by ESIMS, 1 H-, and 2D-NMR. Both compounds showed the same pseudo-molecular-ion peak at m/z 515. The compounds have been previously isolated from the other species in the genus Artemisia such as Artemisia capillaris, Artemisia ciniformis and Artemisia annua (25)(26)(27). The presence of these compounds in the genus Artemisia has led to valuable effects such as anti-giardial, anti-HBV and inhibition of various enzymes like natural protein tyrosine phosphatase 1B , α-Amylase and α-glucosidase and aldose reductase (28)(29)(30)(31)(32). In agreement with the results of the current study, the antioxidant activity of isomers of isochlorogenic acid has been reported in different species of Artemisia (33,34). The results of the Friedman test were consistent with the previous reports for Artemisia biennis which suggests none of three antioxidant assays are significantly different in the selection of active extracts/ fractions (35). In agreement with the previous reports, a significant correlation was observed between the results of the BCB assay and the total phenolic contents (35,36). Because of the very low correlation observed between total phenolic content and FIC results, there should have been some other types of phytochemicals which act as secondary antioxidants in the assay. Nitrogen-containing compounds, terpenoids, and polysaccharide fractions as natural non-phenolic antioxidants are some examples which act with different mechanisms (37)(38)(39)(40)(41). More studies should be conducted to identify and characterize ferrous ion chelating agents of A. turanica.

Conclusion
This study demonstrated the radical scavenging activity of A. turanica, as one of the plant species of Iranian flora. In general, free radical scavenging activities of A. turanica hydroethanolic extract and some of its derived fractions in comparison with other samples could be ascribed to their higher content of phenolic compounds like dicaffeoylquinic acids which were isolated in this study.